Pharmaceutical compositions for treatment of EGF receptor associated cancers

ABSTRACT

The present invention discloses carbohydrates and carbohydrate analogs that bind to epidermal growth factor (EGF) receptors. Methods of using such carbohydrates or analogs for a variety of uses related to the EGF receptor are also provided. In preferred aspects of the present invention, methods for killing or inhibiting the growth of tumor cells with increased EGF receptor activity are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 08/679,643, filed Jul. 12, 1996, now U.S. Pat. No. 6,008,203,which claimed the benefit of U.S. Provisional Application No.60/001,150, filed Jul. 14, 1995, and abandoned.

TECHNICAL FIELD

The present invention relates generally to compounds and methodsassociated with the epidermal growth factor (EGF) receptor. Theinvention is more particularly related to carbohydrates and carbohydrateanalogs that bind to EGF receptors, and methods of use therefor (e.g.,to kill or inhibit the growth of tumor cells with increased EGF receptoractivity).

BACKGROUND OF THE INVENTION

Growth factors are substances that induce cell proliferation, typicallyby binding to specific receptors on cell surfaces. One such growthfactor is epidermal growth factor (EGF). EGF induces proliferation of avariety of cells in vivo, and is required for the growth of mostcultured cells.

The EGF receptor is a 170-180 kD membrane-spanning glycoprotein, whichis detectable on a wide variety of cell types. The extracellularN-terminal domain of the receptor is highly glycosylated and binds EGF.The cytoplasmic C-terminal domain of the receptor contains anEGF-dependent tyrosine-specific protein kinase that is capable of bothautophosphorylation and the phosphorylation of other protein substrates.The two domains are connected by a single 21 amino acid hydrophobictransmembrane region. The binding of EGF to its receptor activates thereceptor tyrosine kinase, which phosphorylates a variety of cellularproteins (including the EGF receptor itself). This phosphorylationinitiates a signal transduction pathway that ultimately leads to DNAreplication, RNA and protein synthesis, and cell division. EGF alsoinduces the concentration of the receptor into clathrin-coated pits,internalization into intracellular vesicles, and finally degradation inthe lysosomes.

The amplification or overexpression of the EGF receptor is associatedwith the uncontrolled cell division of many cancers. Published studiessupport a role for the EGF receptor in cell transformation andmaintenance of the transformed phenotype. There is also a high level ofsequence homology between the EGF receptor and the avian v-erbB oncogeneproduct. In addition, overexpression of the EGF receptor has been shownto result in the EGF-dependent transformation of NIH 3T3 cells.

Many tumors of mesodermal and ectodermal origin overexpress the EGFreceptor. For example, the EGF receptor has been shown to beoverexpressed in many gliomas, squamous cell carcinomas, breastcarcinomas, melanomas, invasive bladder carcinomas and esophagealcancers. In addition, studies with primary human mammary tumors haveshown a correlation between high EGF receptor expression and thepresence of metastases, higher rates of proliferation, and shorterpatient survival.

Attempts to exploit the EGF receptor system for anti-tumor therapy havegenerally involved the use of monoclonal antibodies against the EGFreceptor. However, this approach has serious drawbacks. The monoclonalantibodies developed to date are effective inhibitors of cell growth inonly some of the existing cancer cell lines. In addition, studies withathymic mouse xenografts suggest that therapeutic intervention withanti-receptor antibodies will require prolonged exposure, which mayresult in the generation of antibodies against the anti-receptorantibodies in the patient. Thus, to date, no successful anti-tumortherapies exploiting the EGF receptor system have been developed.

Accordingly, there is a need in the art for improved compounds andmethods for treating EGF-receptor associated cancers. The presentinvention fulfills this need and provides further related advantages.

SUMMARY OF THE INVENTION

Briefly stated, this invention provides compounds and methods related tothe epidermal growth factor (EGF) receptor. In one aspect of theinvention, compounds are provided, having the formula X-Y-Z-R, wherein Xis a sialic acid or a non-saccharide group that mimics the structure ofa sialic acid group, Y and Z are independently a monosaccharide residueor a non-saccharide group that mimics the structure of a monosaccharideresidue, and R is hydrogen or a carrier group, with the proviso thatwhere X is a sialic acid group, Y and Z respectively are not thefollowing: galactose and glucose, galactose and N-acetylglucosamine,galactose and galactose, galactose and N-acetylgalactosamine, sialicacid and sialic acid, sialic acid and galactose, N-acetylgalactosamineand galactose, N-acetylgalactosamine and N-acetylglucosamine, orN-acetylglucosamine and galactose. In an embodiment, X is a sialic acidgroup or a 2-hydroxyacetic acid group, Y is a galactose group or acyclohexane group, Z is a glucose group, a cyclohexane group or anN-acetylglucosamine group, and R is hydrogen or a carrier group, withthe proviso that where X is a sialic acid group and Y is a galactosegroup, then Z is not a glucose group or an N-acetylglucosamine group.

In a related embodiment, the present invention provides compositionscomprising a liposome that includes a compound as described above or acarbohydrate compound having a type 2 saccharide chain without a sialicacid residue or an analog thereof, wherein said compound contains acarrier group. In an embodiment, the compound has the formula LNnT-C,wherein LNnT is a lacto-N-neotetraose group and C is a carrier group.

In another aspect, pharmaceutical compositions are provided, comprising:(a) a compound as described above or a carbohydrate compound having atype 2 saccharide chain without a sialic acid residue or an analogthereof; and (b) a pharmaceutically acceptable carrier or diluent. In anembodiment, the compound has the formula LNnT-C, wherein LNnT is alacto-N-neotetraose group and C is a carrier group.

In another aspect of the invention, methods are provided for purifyingan EGF receptor, comprising: (a) contacting a preparation containing anEGF receptor with a sialylated lactose carbohydrate compound, acarbohydrate compound having a type 2 saccharide chain without a sialicacid residue, or an analog of either, wherein said compound isimmobilized on a solid support, and wherein said contacting takes placeunder conditions such that said EGF receptor binds to said immobilizedcompound; (b) separating the preparation from the immobilized compound,wherein the EGF receptor remains bound to the immobilized compound; and(c) isolating the bound EGF receptor from the immobilized compound.

In a further aspect of this invention, methods are provided forscreening for a candidate molecule able to bind to an EGF receptor,comprising: (a) contacting an EGF receptor immobilized on a solidsupport with a candidate molecule and with a sialylated lactosecarbohydrate compound, a carbohydrate compound having a type 2saccharide chain without a sialic acid residue, or an analog of either;(b) separating unbound compound from the immobilized EGF receptor; and(c) detecting compound bound to the immobilized EGF receptor. In anotherembodiment, the method for screening comprises: (a) contacting acompound immobilized on a solid support with a candidate molecule andwith an EGF receptor, wherein said compound is a sialylated lactosecarbohydrate compound, a carbohydrate compound having a type 2saccharide chain without a sialic acid residue, or an analog of either;(b) separating unbound EGF receptor from said immobilized compound; and(c) detecting EGF receptor bound to said immobilized compound, therefromdetermining whether said candidate molecule binds to said EGF receptor.

In yet another aspect, this invention provides methods for inhibitingEGF receptor kinase activity comprising contacting an EGF receptor witha sialylated lactose carbohydrate compound, a carbohydrate compoundhaving a type 2 saccharide chain without a sialic acid residue, or ananalog of either. In an embodiment, the compound has the formula LNnT-R,wherein LNnT is a lacto-N-neotetraose group and R is hydrogen or acarrier group.

In a related aspect, methods are provided for inhibiting tumor cellgrowth within a biological preparation, comprising contacting abiological preparation with a sialylated lactose carbohydrate compound,a carbohydrate compound having a type 2 saccharide chain without asialic acid residue, or an analog of either. In an embodiment, thecompound has the formula LNnT-R, wherein LNnT is a lacto-N-neotetraosegroup and R is hydrogen or a carrier group.

In a further related aspect, methods are provided for killing tumorcells in a biological preparation, comprising contacting a biologicalpreparation with a sialylated lactose carbohydrate compound, acarbohydrate compound having a type 2 saccharide chain without a sialicacid residue, or an analog of either. In an embodiment, the compound hasthe formula LNnT-C, wherein LNnT is a lacto-N-neotetraose group and C isa carrier group.

In yet another aspect, the present invention provides methods forinhibiting growth, in a warm-blooded animal, of a tumor cell for whichan EGF receptor is associated, comprising administering in an amounteffective to inhibit growth of said tumor cell a sialylated lactosecarbohydrate compound, a carbohydrate compound having a type 2saccharide chain without a sialic acid residue, or an analog of either.In an embodiment, the compound has the formula LNnT-R, wherein LNnT is alacto-N-neotetraose group and R is hydrogen or a carrier group.

In a related aspect, this invention provides methods for killing, in awarm-blooded animal, a tumor cell for which an EGF receptor isassociated, comprising administering in an amount effective to kill saidtumor cell a sialylated lactose carbohydrate compound, a carbohydratecompound having a type 2 saccharide chain without a sialic acid residue,or an analog of either. In an embodiment, the compound has the formulaLNnT-C, wherein LNnT is a lacto-N-neotetraose group and C is a carriergroup.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the inhibition of EGF receptor autophosphorylation by3′ SL-HSA.

FIG. 2 shows the level of inhibition of EGF receptor autophosphorylationby a series of representative carbohydrates of this invention. Lane 1shows the response in the absence of EGF, lane 2 shows the response inthe presence of EGF without carbohydrate, and lanes 3-7 show theresponse in the presence of EGF and one of the following: LNnT-HSA,3′SL-HSA, 6′SL-HSA, LSTa-HSA and 3′SL-HSA (repeat), respectively.

FIG. 3 illustrates the inhibition of EGF receptor autophosphorylation by3′ SL-BSA and 3′ SL-dendrimer. (▾) 3′SL-BSA, (◯) 3′SL-dendrimer, ()BSA.

FIG. 4 shows the inhibition of EGF receptor kinase activity in cellmembranes by 3′ SL-BSA and 3′ SL-dendrimer. ³²P incorporation isrepresented in the absence (Column 1) or presence (Column 2) of EGFwithout added carbohydrate. Columns 3 and 4 show inhibition ofincorporation in the presence of EGF by 3′SL-BSA and 3′SL-dendrimer,respectively.

FIG. 5 illustrates the binding of EGF receptor (EGF-R) to immobilized3′SL-HSA.

FIG. 6 shows the binding of the external domain of EGF receptor toimmobilized neoglycoconjugates.

FIG. 7 illustrates the effect of 3′SL conjugates on the proliferation ofhuman epidermoid carcinoma cells (A1S).

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms to beused hereinafter. In the context of this invention, a “carbohydrate” isa compound containing one or more monosaccharide residues.“Monosaccharides” include unsubstituted, nonhydrolyzable saccharidessuch as glucose or galactose, as well as modified, nonhydrolyzablesaccharides in which one or more hydroxyl groups contain substitutionsor have been replaced with hydrogen or substituted carbon atoms. Onesuch substituted monosaccharide is sialic acid. In the context of thisinvention, an “EGF receptor” includes the entire protein, itsextracellular domain and any portion of the protein able to bind EGF.

Portions of a carbohydrate molecule may consist of non-saccharidegroups. In particular, groups such as cyclohexane that mimic thestructure of a monosaccharide residue may be present. A “carbohydrateanalog,” in the context of this invention, is a compound that has athree dimensional structure similar to that of a carbohydrate, but thatcontains few or no monosaccharide residues. Three dimensional structuresmay be determined using computer modeling programs such as, for example,the model of Hricouini et al., Biochem. 31:10018-10023 (1992). Inparticular, carbohydrates and carbohydrate analogs may contain groupssuch as anti-idiotypic antibodies or cyclohexane derivatives that mimicthe structure of a monosaccharide residue.

Non-saccharide carrier groups may also be present within compounds ofthis invention. A “carrier group” is any group that facilitates thetransport, binding or activity of a compound (i.e., a carbohydrate orcarbohydrate analog). For example, a carrier group may facilitateincorporation of a compound into a cell membrane or liposome, or mayimprove the binding of a compound to an EGF receptor. In anotherexample, a multivalent presentation of a compound may increase thecompound's conjugate exponentially by increasing its avidity for the EGFreceptor. A carrier group may also, or alternatively, kill the cell towhich the compound binds. Such carrier groups include, for example,cytotoxic agents, toxins, radionuclides, and prodrugs. A “conjugate,” inthe context of this invention, is any carbohydrate or carbohydrateanalog that contains a carrier group.

As noted above, the present invention is generally directed to compoundsand methods associated with the EGF receptor (including, for example,killing or inhibiting the growth of tumor cells in EGFreceptor-associated cancers). In one embodiment, the compounds of thepresent invention are members of the sialylated lactose series(including both type 1 and type 2 chains, and including derivatives) oranalogs thereof. Type 1 and type 2 chains refer to polylactosamine unitstructures, i.e., Galβ1→3/4GlcNAc. The polylactosamine having theGalβ1→3GlcNAc structure is called the type 1 chain, and that having theGalβ1→4GlcNAc structure is referred to as the type 2 chain. Examples oftype 1 and type 2 chains are known to those in the art. In anotherembodiment, the compounds useful in this invention are carbohydrateshaving type 2 saccharide chains without a sialic acid residue, includingderivatives thereof such as lacto-N-neotetraose (i.e., LNnT orGalβ1-4GlcNAcβ1-3Galβ1-4Glc). Such compounds may be used, for example,to treat EGF receptor-associated cancers, to purify EGF receptors and toidentify additional EGF receptor-binding compounds.

The compounds of this invention that are members of the sialylatedlactose series (and derivatives) or analogs thereof may be generallyrepresented by the following formula:

X-Y-Z-R

wherein X is a sialic acid group or a non-saccharide group that mimicsthe structure of a sialic acid group, Y and Z represent monosaccharideresidues or non-saccharide groups that mimic the structure of amonosaccharide residue and R is hydrogen or a carrier group. Preferably,X is a sialic acid group or a 2-hydroxyacetic acid group, Y is agalactose group or a cyclohexane group and Z is a glucose group, acyclohexane group or a N-acetylglucosamine group.

In the embodiments in which R is hydrogen, such compounds may generallybe represented by the formula:

X-Y-Z

wherein X, Y and Z are as recited above. Such compounds include, but arenot limited to, 3′-sialyllactose (i.e., Neu5Acα2-3Galβ1-4Glc),6′sialyllactose (i.e., Neu5Acα2-6Galβ1-4Glc) and LSTa (i.e.,Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc), as well as the followingcarbohydrates:

In the embodiments in which R is a carrier group, the members of thesialylated lactose series (including derivatives) or analogs thereof maygenerally be represented by the formula:

X-Y-Z-C

wherein X, Y and Z are as recited above, and C may be any carrier group.In one preferred embodiment, C is a hydrophobic carrier group thatallows incorporation of the compound into a liposome. Such groupsinclude serum albumin (such as bovine serum albumin or human serumalbumin), ceramide, lipids (such as dipalmitoylphosphatidylcholine),cholesterol and long aliphatic chains (e.g., chains containing 8-25carbon atoms in the backbone). In another preferred embodiment, C is anagent capable of killing cells to which the conjugate binds. Forexample, C may be a cytotoxic agent (e.g., daunomycin, doxorubicin,neocarzinostatin or vindesine), a toxin (e.g., ricin A or aribosome-inactivating protein such as saporin), a radionuclide (e.g., aradioactive form of iodine or rhenium), a “prodrug” (i.e., a group thatis not itself toxic to the cell, but that can be rendered toxic afterbinding of the conjugate to the EGF receptor by the addition of a secondactivating compound) or an enzyme that can convert a prodrug into anactive drug. Suitable prodrugs include boron, doxifluridine, or theprodrug precursor of palytoxin. The enzyme may be, for example,beta-lactamase, cytosine deaminase or nitroreductase, which can be usedin conjunction with an appropriate prodrug.

In yet another preferred embodiment, C is an artificial polymer (i.e., apolymer that is not known to be found in nature). Suitable artificialpolymers include polyacrylamide, and derivatives thereof, polylysine anddendrimers (as described, for example, in Tomalia, Scientific American(May, 1995) at 62-66). More preferably, the carrier group is multivalent(i.e., capable of binding to more than one carbohydrate or carbohydrateanalog molecule) or is capable of forming an aggregate structure ofcarrier groups. Such carrier groups have the advantage of increasing thelocal concentration of the non-carrier portion of the carbohydrate. Theabove carrier groups may also be incorporated into carbohydratescontaining a type 2 saccharide chain without a sialic acid residue, suchas lacto-N-neotetraose (LNnT), as described above.

The conjugates of this invention may generally be prepared by coupling acarbohydrate, such as 3′sialyllactose, or a carbohydrate analog to acarrier by any of a variety of means known to those in the art. In onesuch method, a linker such as 2-(4-aminophenyl)ethylamine (APEA) orp-trifluoroacetamidoaniline (which may be synthesized fromp-nitroaniline and trifluoroacetic anhydride, followed by reductivehydrogenation) may be used. Such linkers react with the reducing end ofthe carbohydrate by reductive amination, and may be converted to anisothiocyanate derivative by reacting with thiophosgene. This compoundis highly reactive with amines, and will couple to compounds such asproteins, phosphatidyl ethanolamine and amino groups in dendrimers andpeptides. The use of such linkers has the advantages of simplicity,stability, high yield and compatibility with sialyloligosaccharides.Polylactosamine chains of varying lengths may generally be preparedaccording to the method of Srivastava et al., J. Carbohydrate Chem.10:927-933 (1991), and sialylated according to the method of De Vriesand van den Eijnden, Biochem. 33:9937-9944 (1994). Of course, the abovedescriptions are provided solely for exemplary purposes, and otherlinking methods will be readily apparent to those in the art.

Conjugates of this invention may be incorporated into liposomes.Liposomes are spherical structures that contain, for example, a lipidbilayer surrounding an aqueous interior. The liposome structure ismaintained, in part, by van der Waals interactions between thehydrophobic portions of the lipids. Accordingly, conjugates that containan appropriate hydrophobic carrier group, as described above, may beincorporated into the lipid bilayer of the liposome. A cytotoxic agentor drug may then be encapsulated in the liposome, for delivery to cellsthat contain an EGF receptor. Representative examples of the uses andpharmaceutical compositions of liposomes for cancer chemotherapy aredescribed in Kim, Drugs 46(4):618-638 (1993). In general, incorporationof a conjugate into a liposome may be achieved by combining theconjugate, cholesterol, a lipid (such as dipalmitoylphosphatidylcholine)and a solvent in suitable proportions, and allowing the mixture to dry.Upon the addition of phosphate buffered saline and sonication, liposomesof various sizes are formed. The liposomes may be prepared andsize-selected (e.g., with one or more filters) using any of a variety ofprocedures known to those of ordinary skill in the art, such as (forexample) the methods described in U.S. Pat. Nos. 4,241,046 and4,224,179, and references cited therein.

As noted above, the compounds of this invention may be used for avariety of purposes, including for the purification of EGF receptors,for the screening compounds having the ability to bind to EGF receptorand for the inhibition of EGF receptor kinase activity. Such compoundsmay also be used to inhibit tumor cell growth or to kill tumor cells,either within a biological preparation or in a warm-blooded animal. Theterm “biological preparation,” in the context of this invention,encompasses body samples, cell samples and fractions thereof. Bodysamples include urine, cervical secretions, bronchial aspirates(including bronchial washings), sputum, saliva, feces, serum, umbilicalcord blood, synovial and cerebrospinal fluid, nipple aspirates andascites. Cell samples include tissue samples, as well as cell culturesand suspensions, and cellular components such as membranes. Preferredcell samples are those samples that contain cultured or isolated bonemarrow cells or peripheral blood stem cells or umbilical cord bloodcells.

In one aspect, the compounds of this invention may be used to purify anEGF receptor from any appropriate biological preparation. For example,EGF receptor may be purified from preparations of membrane proteinsobtained from suitable cultured cells, such as A431 cells. EGF receptorthat is purified in this manner may be naturally-occurring orrecombinant.

Typically, purification of EGF receptor may be achieved using acarbohydrate or carbohydrate analog that has been immobilized on a solidsupport. Suitable support materials include, but are not limited to,chromatography support materials, glass beads, polymeric matrices,plastics (such as latex, polystyrene or polyvinylchloride), sinteredglass discs, fiberglass membranes, polymeric membranes and magneticparticles. Magnetic particles may contain a thermoplastic resin combinedwith a magnetically responsive powder, and coated with a polymer havingreactive functional groups. Such particles are disclosed, for example,in U.S. Pat. No. 5,200,270. Alternatively, magnetic particles maycontain a magnetic metal oxide coated with a polymer (such as apolysaccharide or a silane) having pendant functional groups for bindingthe carbohydrate or carbohydrate analog, as disclosed, for example, inU.S. Pat. Nos. 4,452,773; 4,661,408; 4,695,393 and 5,169,754. Theparticles may also have a metal oxide surface (with a metal or polymercore) to which the carbohydrate or carbohydrate analog is directlybound, by the methodology disclosed, for example, in U.S. Pat. Nos.4,018,886 and 5,320,944. Other suitable particles will be apparent tothose of ordinary skill in the art.

The carbohydrate or carbohydrate analog may be immobilized on thesupport using a variety of techniques known to those of ordinary skillin the art. In the context of the present invention, the term“immobilization” refers to both covalent attachment (which may be adirect linkage between the carbohydrate or carbohydrate analog andfunctional groups on the support, or which may be a linkage by way of across-linking agent) and noncovalent association, such as adsorption.Briefly, covalent attachment may be achieved by first reacting thesupport with a bifunctional reagent (see, e.g., catalog of PierceChemical Co., Rockford, Ill.), such as benzoquinone, that will reactwith both the support and a functional group, generally a hydroxylgroup, on the carbohydrate or carbohydrate analog. Alternatively, thecarbohydrate or carbohydrate analog may be immobilized on a support bycondensation of an aldehyde group on the support with an amine and anactive hydrogen on the carbohydrate or analog (e.g., in a Mannichreaction).

The carbohydrate or carbohydrate analog is preferably immobilized byadsorption to a support, using any of a variety of techniques. Forexample, a conjugate that includes a suitable carrier, such as bovineserum albumin (BSA) or the constant portion of the heavy chain of gammaimmunoglobulin, may be physically adsorbed to appropriate surfaces ofparticles or other solid supports by contacting the conjugate with thesupport for a suitable amount of time. The contact time varies withtemperature, but is typically between about 1 hour and 1 day. Followingadsorption, the coated support is typically incubated with a blockingagent, such as bovine serum albumin in phosphate buffered saline, toblock non-specific binding to the support.

The immobilized conjugate may then be contacted with the preparationcontaining EGF receptor for an incubation time and under conditions thatpermit binding of the EGF receptor to the immobilized conjugate.Suitable conditions will be apparent to those of ordinary skill in theart. In general, physiological conditions (e.g., phosphate-bufferedsaline) are appropriate.

After incubation, the support to which EGF is bound may be separatedfrom the remainder of the preparation and washed to remove contaminants.The substantially pure EGF receptor may then be removed from the supportin a suitable eluent, and analyzed to confirm the presence of active EGFreceptor. Such analyses may be performed using a variety of techniquesknown to those of ordinary skill in the art, including the method ofKing et al., Life Sciences 53:1465-1472 (1993). EGF receptor purified inthis manner may be detected as a single 170-180 kD band using SDS-PAGE.No other kinase activity is detectable (i.e., no other phosphorylatedproteins are detected).

The compounds of this invention may also be used to screen candidatemolecules for the ability to bind to EGF receptor. In one embodiment,the screen is performed using a competition assay. Briefly, in thisembodiment, EGF receptor is immobilized on a support and incubated withboth the candidate molecule and a carbohydrate or carbohydrate analog.After incubation, the unbound compounds are removed, and the boundcarbohydrate or carbohydrate analog is detected using a reporter group.Typical reporter groups include radioisotopes (such as tritium),fluorescent groups, luminescent groups, enzymes (such as horseradishperoxidase or alkaline phosphatase), substrates, cofactors, inhibitors,biotin and dye particles. The reporter group may be linked to thecarbohydrate or carbohydrate analog, or to a separate molecule thatbinds to the carbohydrate or analog. In either case, the amount of boundreporter group detected is inversely related to the amount of boundcandidate molecule (i.e., a lower amount of bound reporter groupindicates that the candidate molecule has a greater ability to bind tothe EGF receptor).

In general, EGF receptor may be immobilized directly on any appropriatesupport, such as the well of a microtiter plate or other supportdescribed above. Alternatively, an anti-EGF receptor antibody (which maybe prepared using techniques well known to those of ordinary skill inthe art) may be immobilized on the support, and EGF receptor may bebound to the immobilized antibody. Immobilization of EGF receptor oranti-EGF receptor antibody may generally be achieved by the methodsdescribed above for the immobilization of carbohydrates.

The carbohydrate or carbohydrate analog employed in this method may, butneed not, contain a carrier group. In addition, as discussed above, thecarbohydrate or analog may contain a reporter group. Reporter groups maybe incorporated by any of a variety of techniques known to those in theart (see, e.g., catalog of Pierce Chemical Co., Rockford, Ill.). Forexample, tritium may be incorporated using sodium borohydride (see,e.g., catalog of Oxford GlycoSystems, Rosedale, N.Y.). Reporter groupsmay also be linked via a spacer arm (e.g., APD or APEA) that is joinedto the carbohydrate or carbohydrate analog by reductive amination.

The immobilized EGF receptor is incubated with the candidate moleculeand the carbohydrate or carbohydrate analog to allow the bindingreactions to approach equilibrium. An optimal incubation time may bedetermined by measuring the time dependence of binding, and selecting atime at which an appropriate percentage (e.g., 95%) of maximum bindinghas been achieved. Unbound molecules are then removed from the supportby separation (e.g., by filtration), optionally followed by anappropriate wash. If the carbohydrate or carbohydrate analog contains areporter group, the reporter group may then be detected. Radioactivereporter groups may be detected using, for example, scintillationcounting. Fluorescent groups may be detected using, for example,fluorometric techniques. Spectroscopic methods may be used to detectdyes and luminescent groups. Biotin may be detected using, for example,streptavidin coupled to a reporter group (commonly a radioactive orfluorescent group or an enzyme). Enzyme reporter groups may generally bedetected by addition of substrate (generally for a specific period oftime), followed by spectroscopic or other analysis of the reactionproducts.

Alternatively, if the reporter group is bound to a molecule (such as anantibody, lectin or other carbohydrate binding protein) that is capableof binding to the carbohydrate or carbohydrate analog, the moleculecontaining the reporter group may be added to the immobilized EGFreceptor after removal of the unbound carbohydrate or carbohydrateanalog, and incubated for an appropriate period of time. Suitablereporter groups for antibodies include radioisotopes, such asradioactive iodine, and other groups described above. The incubationtime may be optimized by evaluating the time dependence of binding.After incubation, the unbound antibody is removed prior to detection ofthe reporter group, as described above. In this manner, the boundcarbohydrate or carbohydrate analog may be detected indirectly, based onthe level of the bound antibody.

In another embodiment of the screening for candidate molecules, avariation on the competition assay described above may be used. It willbe evident to those in the art that portions of the above discussion areapplicable to this embodiment as well. Briefly, in this embodiment, acarbohydrate compound or analog thereof is immobilized on a support andincubated with both the candidate molecule and EGF receptor. Afterincubation, the unbound EGF receptor is removed, and the bound EGFreceptor is detected. For example, an antibody which is against EGFreceptor and which possesses a reporter group may be used.Alternatively, a second antibody which is against the anti-EGF receptorantibody and which contains a reporter group may be used. Other examplesof alternative detection methodologies are described above.

In another embodiment, the screen for candidate molecules having theability to bind to an EGF receptor is a direct assay. In thisembodiment, the molecule to be tested is immobilized on a support, suchas the well of a microtiter plate, as described above. EGF receptor(which may be purified as described above) is then allowed to bind tothe immobilized candidate molecule. The EGF receptor may be linked to areporter group, or may be detected using an anti-EGF receptor antibodyto which a reporter group is linked, as described above. In thisembodiment, a higher level of reporter group detected indicates amolecule with a greater ability to bind to an EGF receptor.

Within other aspects, the compounds of this invention may be used toinhibit EGF receptor kinase activity, to inhibit tumor cell growthand/or to kill tumor cells in biological preparations or in warm-bloodedanimals, preferably humans. Suitable biological preparations forinhibiting EGF receptor kinase activity are any preparations thatcontain EGF receptors (e.g., preparations of membrane proteins from celllines such as A431 (ATCC No. CRL 1555), U118-MG (ATCC No. HTB15), and/orU373-MG (ATCC No. HTB17)). For inhibiting tumor cell growth or killingtumor cells, suitable biological preparations and warm-blooded animalsare those that contain tumor cells that express EGF receptor (i.e.,tumor cells with which EGF receptor is associated).

In one such aspect, inhibition of kinase activity may generally beachieved by contacting an appropriate amount of a compound as describedabove with EGF receptor, either in vitro or in vivo. For example, thekinase activity of EGF receptor within a biological preparation may beinhibited by combining a suitable amount of carbohydrate or carbohydrateanalog with the biological preparation. Kinase activity may be measuredby incubating the EGF receptor with a test peptide (such as the RR-srcpeptide, which contains a single tyrosine residue) and radiolabeled ATP,and measuring the amount of label incorporated into the test peptideusing, for example, a liquid scintillation counter. Alternatively,autophosphorylation of the EGF receptor in intact cells may be measuredwith anti-phosphotyrosine antibody, as described in Arvidsson et al.,Mol. Cell. Biol. 14:6715 (1994) or Glenney et al., J. Immun. Meth.109:277 (1988). Kinase activity of EGF receptor in a warm-blooded animalmay generally be inhibited by administration of an effective amount ofthe carbohydrate or carbohydrate analog to the animal.

For inhibiting kinase activity, without killing the target cell, thecompound may be any of the carbohydrates or carbohydrate analogsdescribed above. In preferred embodiments, the compound is a conjugate.However, if cell survival is desired, the use of carrier groups thatkill the target cell should be avoided.

For administration to warm-blooded animals, the carbohydrate orcarbohydrate analog may be combined with a pharmaceutically acceptablecarrier or diluent to form a pharmaceutical composition. Any of avariety of carriers or diluents may be employed. Liquid diluentsinclude, for example, water, saline, dextrose, glycerol, ethanol andcombinations thereof. For solid pharmaceutical compositions, suitablecarriers include pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, cellulose, magnesium carbonate and the like.Pharmaceutical compositions may also contain one or more binders (suchas gelatin, acacia or corn starch), sweeteners (e.g., lactose, sucroseor saccharin), flavoring agents (e.g., oil of wintergreen, peppermint orfruit flavors), lubricants (e.g., magnesium stearate) and/or excipients(e.g., dicalcium phosphate). The pharmaceutical composition may take avariety of forms, including tablets, capsules, powders, granules,emulsions, ointments, gels, foams or aerosols. In addition, thecarbohydrate in the pharmaceutical composition may be incorporated intoa liposome. Administration may be by any means sufficient to expose thedesired region of the animal to the carbohydrate or carbohydrate analog,including oral administration, inhalation, and injection (e.g.,intravenous, intracutaneous, intramuscular or subcutaneous).

An effective amount of carbohydrate or carbohydrate analog may bedetermined using methods known to those of ordinary skill in the art.Specifically, dose response curves may be used to determine an effectiveamount for inhibiting EGF receptor activity within a biologicalpreparation, preferably without causing significant cell death. Doseresponse curves may also be generated in animals. An appropriate amountfor treatment of humans may be determined by extrapolating from animaldata or in vitro data, and/or using clinical trials. Suitable doses andcourses of treatment depend upon a variety of factors, including thespecific compound used, the size of the patient and the method ofadministration. A suitable amount of the compound for administration toa warm-blooded animal will be up to and including the highesttherapeutic dose that does not cause unacceptable side effects ortoxicity (as determined in animal models, followed by clinical trials).In general, a suitable dose of carbohydrate or carbohydrate analog is anamount sufficient to raise the blood concentration of carbohydrate orcarbohydrate analog to 10⁻¹⁵ to 10⁻² M. Doses may be administered onceor several times per day.

To inhibit tumor cell growth in a biological preparation or awarm-blooded animal, an appropriate amount of a compound as describedabove is contacted with tumor cells containing EGF receptors. Tumor cellgrowth in a biological preparation may be inhibited by incubating anappropriate amount of the compound with the biological preparation.Similarly, the growth of in vivo tumors may generally be inhibited byadministering an effective amount of the compound to the affectedwarm-blooded animal by any of the methods described above.

If, in this aspect, a conjugate is employed, and cell survival isdesired, any of the above carriers that are not lethal to the targetcell may be employed. Suitable amounts of carbohydrate or carbohydrateanalog may generally be determined, as described above, using doseresponse curves and by evaluating the time dependence of the response.For tumors in a warm-blooded animal, the compound generally may beadministered to the animal as discussed above.

The compounds of this invention may also be used to kill tumor cells ina biological preparation or in a warm-blooded animal. In one embodiment,a conjugate is employed that contains a carrier group capable of killingthe cell, such as a cytotoxic agent, a toxin, a radionuclide (e.g.,radioactive iodine), a prodrug, or an enzyme that converts a prodrug toan active drug, as described above. Alternatively, a conjugate may beincorporated into a liposome, as described above, and the cytotoxicagent, prodrug or other agent capable of killing the cell may beencapsulated in the liposome for delivery to the tumor cell. Anyappropriate agent known to those of ordinary skill in the art may beused for this purpose.

Tumor cells in a biological preparation may be killed by incubating theconjugate with the biological preparation. A suitable amount ofconjugate and an appropriate incubation time may generally bedetermined, as described above, using dose response curves and byevaluating the time dependence of the response.

In this embodiment, the biological preparation preferably contains bonemarrow cells, and the incubation takes place prior to autologous bonemarrow transplantation. Bone marrow cells may be isolated by any of avariety of techniques known to those of ordinary skill in the art. Forexample, commercially available kits, such as CEPRATE (CellPro, Inc.,Bothell, Wash.) may be used according to the manufacturer'sinstructions. The bone marrow cells are incubated with the conjugate foran amount of time sufficient to kill the cancer cells, but underconditions that will maintain the viability and proliferative potentialof the bone marrow cells. Such conditions may be determined by doseresponse curves, as discussed above. For killing tumor cells in awarm-blooded animal, the conjugate is preferably administered in theform of a pharmaceutical composition, as described above.

A sialylated lactose carbohydrate compound, a carbohydrate compoundhaving a type 2 saccharide chain without a sialic acid residue, or ananalog of either, may be used alone or in combination (e.g., with oneanother and/or with one or more other compounds) in the preparation ormanufacture of a medicament. Such medicaments may be for a variety ofuses, including for the inhibition of tumor cell growth or for killingtumor cells (e.g., where a tumor cell has increased EGF receptoractivity) such as for the treatment of EGF receptor associated cancers.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Synthesis of Carbohydrate Conjugates

This Example illustrates the synthesis of carbohydrates and carbohydrateanalog conjugates.

A. Conjugation to Serum Albumin via APD

Carbohydrates and carbohydrate analogs may be coupled to human serumalbumin by way of aminophenylene diamine (APD) as described below for3′-sialyllactose (3′SL). The carbohydrate portions of these conjugatesmay be obtained from commercial sources such as GlycoTech Corp.,Gaithersburg, Md. or Oxford GlycoSystems, Rosedale, N.Y.

The APD linker arm is derived from p-trifluoroacetamidoaniline (TFAN),which is synthesized from 4-trifluoroacetamidonitrobenzene. 2.76 g ofp-nitroaniline is dissolved in 25 ml of redistilled pyridine, and thedark yellow solution is cooled in an ice bath. 5 ml of trifluoroaceticanhydride is added dropwise to the solution under constant stirring. Thesolution is then filtered on a sintered glass filter and washed withcold water. The solid material is recrystallized by adding approximately100 ml of ethanol and gradually adding an additional 50 ml of ethanolwhile heating to 60° C. under stirring. The solution is covered andplaced in the refrigerator overnight for recrystallization.

The solution is refiltered on a sintered glass filter, and the crystalsare washed with cold water and dried under high vacuum. Yield of4-trifluoroacetamidonitrobenzene is about 90-92% and the melting pointis 140° C. The crystals are stored under desiccation at −20° C. orimmediately subjected to reductive hydrogenation.

Reductive hydrogenation is achieved by adding 2 grams of4-trifluoroacetamidonitrobenzene to 50 mg of palladium on activatedcharcoal in 30 ml of ethanol. Hydrogen gas is bubbled in the solution atroom temperature for 3 hr. The solution is then filtered on Whatman No.1 paper to remove the charcoal. The filter is washed with ethanol andthe filtrate is pooled with the washing and evaporated to dryness byrotary evaporation. The product is crystallized by dissolving theresidue in 20-40 ml of ethyl ether. Hexane is gradually added and thelight pink crystals are collected by filtration and washed with hexane.The resulting 4-trifluoroacetamidoaniline (TFAN) has a melting point of118° C. and a yield of 76%.

TFAN is used to couple oligosaccharides such as 3′sialyllactose toamine-containing proteins such as serum albumin. In the first step ofthe reaction, the oligosaccharide is coupled to TFAN by reductiveamination followed by acetylation. 2 mg of 3′sialyllactose is added to10 mg of TFAN in 0.3 ml of ethanol/water 2:1 (v/v). 0.02 ml of glacialacetic acid and 6 mg of NaCNBH₃ is added under stirring. The glass vialis capped and incubated under stirring at 30° C. for 15-20 hr. Thereaction is monitored by TLC, as described below.

0.05 ml of acetic anhydride is added and incubated at 30° C. for anadditional 3 hr. The reaction is monitored for complete acetylation byTLC. The reaction is then transferred to a 13×100 mm glass test tubewith a PTFE lined screw cap. 5 ml of ethyl acetate is added, followed by5 ml of water for extraction. The phases are separated and the waterphase is washed with another 5 ml of ethyl acetate. The water phases arecombined and concentrated to 1-2 ml by rotary evaporation.

The concentrate is applied to a C₁₈ column (4×0.4 mm i.d.)preconditioned with methanol followed by water. After washing with 5column volumes of water, the N-acetylated TFAN derivative of3′sialyllactose is eluted with 5 column volumes of methanol. The sampleis evaporated to dryness by rotary evaporation.

The second step of the reaction removes the trifluoro group from thenitrogen. The dry TFAN derivative is dissolved in 0.5 ml of 0.5 M NaOHand incubated at room temperature for 3 hr. The reaction is monitored byTLC.

The third step of the reaction converts the amine to an isothiocyanate.1 ml of ethanol is added to the reaction, followed by 0.02 ml glacialacetic acid. The pH is adjusted to about 7 with 0.1 ml of 0.2 M NaOH.Under constant stirring, 0.004 ml of thiophosgene is carefully added.The pH is adjusted to above 5 with another 0.1 ml of 0.2 M NaOH.Conversion of the aromatic amine to the isothiocyanate is almostinstantaneous.

The reaction product is extracted with 0.5 ml water and 1 ml ethylether. The upper phase (ether) is washed again with 0.5 ml water. Thewater phases are combined and rotary evaporated to 0.5-1 ml.

The last step of the reaction couples the isothiocyanate derivative tothe amine-containing carrier molecule, such as serum albumin. Theisothiocyanate derivative is transferred to a 5 ml vial containing 5 mgof human serum albumin in 0.1 M borate buffer at pH 9.4. The pH of thesolution is readjusted to 9.4 with 0.5 M NaOH. The reaction is incubatedunder constant stirring at room temperature for at least 20 hr, and ismonitored by TLC. The reaction is then dialyzed against three changes of20 mM NH₄HCO₃, lyophilized and the degree of coupling is characterizedby using standard assays for protein and sialic acid.

The TLC analyses described above are performed on silica gel 60 HPTLCthin layer plates in ethyl acetate/methanol/acetic acid/water, 6:3:3:2(v/v/v/v). The Rf values are as follows:

Compound Rf Value 3′SL 0.22 3′SL-TAD 0.43 3′SL-NAc-TAD 0.39 TAD 1.003′SL-NAc-aniline 0.08 3′SL-NAc-Ph-N═C═S 0.66

B. Conjugation to Serum Albumin via APEA

APEA is coupled to carbohydrates and carbohydrate analogs by reductiveamination, as described below for 3′-sialyllactose (3′SL). 5 mg of NaBH₄and 2 mg of 3′sialyllactose are added to 0.25 ml APEA and incubatedunder stirring for 16 hr at 35° C. Excess reagent is destroyed by addingacetic acid to lower the pH to 5.6 in an ice bath. The sample isevaporated to dryness by rotary evaporation.

The sample is dissolved in 0.1 M pyridine/acetate buffer, pH 5.0 andchromatographed in the same buffer on BioGel P-2. The fractions that areboth UV adsorbent and orcinol positive are combined and evaporated todryness. The sample is dissolved in water and passed through a C18column. The product (3′sialyllactose-APEA) is not retained by the columnand is collected.

0.73 mg of 3′sialyllactose-APEA is added to 0.2 ml of 0.5 M NaAc and 0.4ml ethanol. While stirring carefully, 1.6 μl of thiophosgene is added.Neutral pH is maintained by the addition of 0.2 M NaOH. After 10 min.,the mixture is evaporated to near dryness and reconstituted with 0.8 mlof 0.1 M borate buffer, pH 9.5. Synthesis of the isothiocyanatederivative is monitored by TLC as described below.

0.2 mg of serum albumin is added directly to the3′sialyllactose-APEA-isothiocyanate derivative. The reaction isincubated at room temperature under constant stirring for 48 hr andmonitored by TLC. The 3′sialyllactose-albumin containing solution isthen dialyzed against an appropriate buffer.

The TLC analyses are performed as described above, with the following Rfvalues:

Compound Rf Value 3′SLac 0.22 3′SLac-APEA 0.0573′SLac-APEA-isothiocyanate 0.55 3′SLac-APEA-albumin 0.00

C. Conjugation to Dendrimer Carrier Groups via APD

Carbohydrates and carbohydrate analogs may be coupled to generation 4dendrimers via APD as described below for 3′sialyllactose. Thecarbohydrate portions of these conjugates may be obtained fromcommercial sources such as Oxford GlycoSystems, Rosedale, N.Y.

The APD linker arm is derived from p-trifluoroacetamidoaniline (TFAN),which is synthesized from 4-trifluoroacetamidonitrobenzene. 2.76 g ofp-nitroaniline is dissolved in 25 ml of redistilled pyridine, and thedark yellow solution is cooled in an ice bath. 5 ml of trifluoroaceticanhydride is added dropwise to the solution under constant stirring. Thesolution is then filtered on a sintered glass filter and washed withcold water. The solid material is recrystallized by adding approximately100 ml of ethanol and gradually adding an additional 50 ml of ethanolwhile heating to 60° C. under stirring. The solution is covered andplaced in the refrigerator overnight for recrystallization.

The solution is refiltered on a sintered glass filter, and the crystalsare washed with cold water and dried under high vacuum. Yield of4-trifluoroacetamidonitrobenzene is about 90-92% and the melting pointis 140° C. The crystals are stored under desiccation at −20° C. orimmediately subjected to reductive hydrogenation.

Reductive hydrogenation is achieved by adding 2 grams of4-trifluoroacetamidonitrobenzene to 50 mg of palladium on activatedcharcoal in 30 ml of ethanol. Hydrogen gas is bubbled in the solution atroom temperature for 3 hr. The solution is then filtered on Whatman No.1 paper to remove the charcoal. The filter is washed with ethanol andthe filtrate is pooled with the washing and evaporated to dryness byrotary evaporation. The product is crystallized by dissolving theresidue in 20-40 ml of ethyl ether. Hexane is gradually added and thelight pink crystals are collected by filtration and washed with hexane.The resulting 4-trifluoroacetamidoaniline (TFAN) has a melting point of118° C. and a yield of 76%.

TFAN is then used to couple the 3′sialyllactose to a dendrimer. In thefirst step of the reaction, the oligosaccharide is coupled to TFAN byreductive amination followed by acetylation. 2 mg of 3′sialyllactose isadded to 10 mg of TFAN in 0.3 ml of ethanol/water 2:1 (v/v). 0.02 ml ofglacial acetic acid and 6 mg of NaCNBH₃ is added under stirring. Theglass vial is capped and incubated under stirring at 30° C. for 15-20hr. The reaction is monitored by TLC, as described below.

0.05 ml of acetic anhydride is added and incubated at 30° C. for anadditional 3 hr. The reaction is monitored for complete acetylation byTLC. The reaction is then transferred to a 13×100 mm glass test tubewith a PTFE lined screw cap. 5 ml of ethyl acetate is added, followed by5 ml of water for extraction. The phases are separated and the waterphase is washed with another 5 ml of ethyl acetate. The water phases arecombined and concentrated to 1-2 ml by rotary evaporation.

The concentrate is applied to a C₁₈ column (4×0.4 mm i.d.)preconditioned with methanol followed by water. After washing with 5column volumes of water, the N-acetylated TFAN derivative of3′sialyllactose is eluted with 5 column volumes of methanol. The sampleis evaporated to dryness by rotary evaporation.

The second step of the reaction removes the trifluoro group from thenitrogen. The dry TFAN derivative is dissolved in 0.5 ml of 0.5 M NaOHand incubated at room temperature for 3 hr. The reaction is monitored byTLC.

The third step of the reaction converts the amine to an isothiocyanate.1 ml of ethanol is added to the reaction, followed by 0.02 ml glacialacetic acid. The pH is adjusted to about 7 with 0.1 ml of 0.2 M NaOH.Under constant stirring, 0.004 ml of thiophosgene is carefully added.The pH is adjusted to above 5 with another 0.1 ml of 0.2 M NaOH.Conversion of the aromatic amine to the isothiocyanate is almostinstantaneous.

The reaction product is extracted with 0.5 ml water and 1 ml ethylether. The upper phase (ether) is washed again with 0.5 ml water. Thewater phases are combined and rotary evaporated to 0.5-1 ml.

The last step of the reaction couples the isothiocyanate derivative tothe dendrimer. The isothiocyanate derivative is transferred to a 5 mlvial containing an amount of a generation 4 dendrimer (Aldrich ChemicalCo., Milwaukee, Wis.) in 0.1 M borate buffer at pH 9.4 sufficient toprepare a glycoconjugate with the desired mole ratio of carbohydrate todendrimer. The pH of the solution is readjusted to 9.4 with 0.5 M NaOH.The reaction is incubated under constant stirring at room temperaturefor at least 20 hr, and is monitored by TLC. The reaction is thendialyzed against three changes of 20 mM NH₄HCO₃, lyophilized and thedegree of coupling is characterized using standard assays for proteinand sialic acid.

D. Conjugation to Dendrimer Carrier Groups via APEA

APEA is coupled to carbohydrates and carbohydrate analogs by reductiveamination, as described below for 3′-sialyllactose (3′SL). 5 mg of NaBH₄and 2 mg of 3′sialyllactose are added to 0.25 ml APEA and incubatedunder stirring for 16 hr at 35° C. Excess reagent is destroyed by addingacetic acid to lower the pH to 5.6 in an ice bath. The sample isevaporated to dryness by rotary evaporation.

The sample is dissolved in 0.1 M pyridine/acetate buffer, pH 5.0 andchromatographed in the same buffer on BioGel P-2. The fractions that areboth UV adsorbent and orcinol positive are combined and evaporated todryness. The sample is dissolved in water and passed through a C18column. The product (3′sialyllactose-APEA) is not retained by the columnand is collected.

0.73 mg of 3′sialyllactose-APEA is added to 0.2 ml of 0.5 M NaAc and 0.4ml ethanol. While stirring carefully, 1.6 μl of thiophosgene is added.Neutral pH is maintained by the addition of 0.2 M NaOH. After 10 min.,the mixture is evaporated to near dryness and reconstituted with 0.8 mlof 0.1 M borate buffer, pH 9.5. Synthesis of the isothiocyanatederivative is monitored by TLC as described below.

Generation 4 dendrimer is added directly to the3′sialyllactose-APEA-isothiocyanate derivative in an amount sufficientto prepare a glycoconjugate with the desired mole ratio of carbohydrateto dendrimer. The reaction is incubated at room temperature underconstant stirring for 48 hr and monitored by TLC. The3′sialyllactose-dendrimer containing solution is then dialyzed againstan appropriate buffer.

Example 2 Purification of EGF Receptor

This example illustrates the purification of EGF receptors from A431cell membranes.

A. Preparation of Cell Membranes

Membranes were prepared by scraping A431 cells (American Type CultureCollection, Rockville, Md.) from 150 mm dishes of confluent cells in thepresence of phosphate-buffered saline (PBS). The scraped cells werepelleted by centrifugation at 800 g and resuspended in 6 ml of 5 mMHEPES, pH 7.4, 5 mM MgCl₂, and 5 mM of β-mercaptoethanol. The cells werethen homogenized with 30-50 strokes in a Dounce homogenizer using atight-fitting pestle. 2.1 ml of 1 M sucrose was added to the homogenateand the homogenate was centrifuged at 1200 g for 10 minutes. Thesupernatant was then transferred to a polycarbonate tube and centrifugedat 100,000 g for 1 hr. The pellet was resuspended in 300 μl of 20 mMHEPES, pH 7.4, 100 mM NaCl, 5 MM MgCl₂. Protein concentration wasdetermined as described in Peterson, Anal. Biochem. 83:346 (1977).

B. Purification of EGF Receptor from Cell Membranes

Carbohydrates such as 3′ sialyllactose and carbohydrate analogs may becoupled to solid supports containing free amines, such as PharmaLink Gel(trademark of Pierce Chemical Co., Rockford, Ill.) or by way ofaminophenylene diamine (APD) as described below for 3′-sialyllactose(3′SL). The carbohydrate portions of these conjugates may be obtainedfrom commercial sources such as GlycoTech Corp., Gaithersburg, Md., orOxford GlycoSystems, Rosedale, N.Y.

The APD linker arm is derived from p-trifluoroacetamidoaniline (TFAN),which is synthesized from 4-trifluoroacetamidonitrobenzene. 2.76 g ofp-nitroaniline is dissolved in 25 ml of redistilled pyridine, and thedark yellow solution is cooled in an ice bath. 5 ml of trifluoroaceticanhydride is added dropwise to the solution under constant stirring. Thesolution is then filtered on a sintered glass filter and washed withcold water. The solid material is recrystallized by adding approximately100 ml of ethanol and gradually adding an additional 50 ml of ethanolwhile heating to 60° C. under stirring. The solution is covered andplaced in the refrigerator overnight for recrystallization.

The solution is refiltered on a sintered glass filter, and the crystalsare washed with cold water and dried under high vacuum. Yield of4-trifluoroacetamidonitrobenezene is about 90-92% and the melting pointis 140° C. The crystals are stored under desiccation at −20° C. orimmediately subjected to reductive hydrogenation.

Reductive hydrogenation is achieved by adding 2 grams of4-trifluoroacetamidonitrobenezene to 50 mg of palladium on activatedcharcoal in 30 ml of ethanol. Hydrogen gas is bubbled in the solution atroom temperature for 3 hr. The solution is then filtered on Whatman No.1 paper to remove the charcoal. The filter is washed with ethanol andthe filtrate is pooled with the washing and evaporated to dryness byrotary evaporation. The product is crystallized by dissolving theresidue in 20-40 ml of ethyl ether. Hexane is gradually added and thelight pink crystals are collected by filtration and washed with hexane.The resulting 4-trifluoroacetamidoaniline (TFAN) has a melting point of118° C. and a yield of 76%.

TFAN is used to couple oligosaccharides such as 3′sialyllactose toamine-containing proteins such as serum albumin. In the first step ofthe reaction, the oligosaccharide is coupled to TFAN by reductiveamination followed by acetylation. 2 mg of 3′sialyllactose is added to10 mg of TFAN in 0.3 ml of ethanol/water 2:1 (v/v). 0.02 ml of glacialacetic acid and 6 mg of NaCNBH₃ is added under stirring. The glass vialis capped and incubated under stirring at 30° C. for 15-20 hr. Thereaction is monitored by TLC, as described below.

0.05 ml of acetic anhydride is added and incubated at 30° C. for anadditional 3 hr. The reaction is monitored for complete acetylation byTLC. The reaction is then transferred to a 13×100 mm glass test tubewith a PTFE lined screw cap. 5 ml of ethyl acetate is added, followed by5 ml of water for extraction. The phases are separated and the waterphase is washed with another 5 ml of ethyl acetate. The water phases arecombined and concentrated to 1-2 ml by rotary evaporation.

The concentrate is applied to a C₁₈ column (4×0.4 mm i.d.)preconditioned with methanol followed by water. After washing with 5column volumes of water, the N-acetylated TFAN derivative of3′sialyllactose is eluted with 5 column volumes of methanol. The same isevaporated by dryness by rotary evaporation.

The second step of the reaction removes the trifluoro group from thenitrogen. The dry TFAN derivative is dissolved in 0.5 ml of 0.5 M NaOHand incubated at room temperature for 3 hr. The reaction is monitored byTLC.

The third step of the reaction converts the amine to an isothiocyanate.1 ml of ethanol is added to the reaction, followed by 0.02 ml glacialacetic acid. The pH is adjusted to about 7 with 0.1 ml of 0.2 M NaOH.Under constant stirring, 0.004 ml of thiophosgene is carefully added.The pH is adjusted to above 5 with another 0.1 ml of 0.2 M NaOH.Conversion of the aromatic amine to the isothiocyanate is almostinstantaneous.

The reaction product is extracted with 0.5 ml water and 1 ml ethylether. The upper phase (ether) is washed again with 0.5 ml water. Thewater phases are combined and rotary evaporated to 0.5-1 ml.

The last step of the reaction couples the isothiocyanate derivative tothe amine containing solid support, such as PharmaLink Gel (PierceChemical Co., Rockford, Ill.). The isothiocyanate derivative istransferred to a 5 ml vial containing 25 mg PharmaLink gel in 0.1 Mborate buffer at pH 9.4. The pH of the solution is readjusted to 9.4with 0.5 M NaOH. The reaction is incubated under constant stirring atroom temperature for at least 20 hr, and is monitored by TLC of theliquid phase. The solid support is then removed by filtration orcentrifugation and washed three times with 20 mM NH₄HCO₃ and three timeswith PBS.

The resulting carbohydrate immobilized on a solid support is incubatedwith 0.5 to 1.0 mg of the proteins from an A431 cell lysate for 16 hr at4° C. The solid support is then washed three times with PBS and twicewith PBS containing 0.05% Triton X-100 and 10% glycerol. If the materialis to be used directly in a kinase assay, it is washed one time withphosphorylation buffer. If the EGF receptor is to be eluted from thesolid support, it is washed with a sufficient concentration of free 3′sialyllactose (e.g., 0.5 M) in PBS containing 0.05% Triton X-100 and 10%glycerol to elute the EGF receptor. The protein is then dialysed againstPBS containing 0.05% Triton X-100 and 10% glycerol to remove the 3′sialyllactose.

Example 3 Competition Assay for Binding to EGF Receptor

This example illustrates a competition assay for the binding of acandidate compound to an EGF receptor.

A chimeric protein containing the extracellular portion of the EGFreceptor fused to the constant portion of the heavy chain of γimmunoglobulin is prepared according to the method of Aruffo et al.,Cell 61:1303-1313 (1990).

The chimeric protein (0.2 μg in 100 μl of PBS) is coated on a well of aFalcon Pro-Bind 96 well assay plate (Becton Dickinson, Lincoln Park,N.J.) by incubation at 4° C. overnight or by incubation at 37° C. for 2hr. After this incubation, non-specific binding may be blocked byincubating the coated support with 3% bovine serum albumin in PBS for atleast 1 hr at room temperature, followed by washing four times with PBS.

The immobilized chimeric protein is then incubated with 60 μl of 10 mMcandidate compound (in PBS with 1% BSA) for 2 hr at room temperature. 60μl of 1 μg/mL 3′-sialyllactose conjugated to a biotinylated BSA is thenadded to the mixture, and incubated with the immobilized chimericprotein for an additional 2 hr. The solid support is then washed withPBS, as before.

Bound 3′-sialyllactose is then detected using streptavidin, conjugatedto horseradish peroxidase (Amersham, Arlington Heights, Ill.), accordingto the manufacturer's instructions. After incubation with streptavidin,the solid support is washed four times with PBS.

The presence of horseradish peroxidase is quantified by adding hydrogenperoxide and TMB, a substrate for HRP, stopping the reaction by theaddition of 1 M H₃PO₄, and measuring the absorbance at 450 nm. Candidatecompounds that generate a signal that is at least 50% of the signalachieved with 3′SL are considered to bind to EGF receptor.

Example 4 Direct Assay for Binding to EGF Receptor

This example illustrates a direct assay for identifying compounds thatbind to the EGF receptor.

Compounds to be tested are allowed to bind to wells in a 96-wellmicrotiter plate (e.g., Probind, Falcon, Lincoln Park, N.J.) or to PVDFmembranes. The compounds are immobilized on the wells by incubation at4° C. overnight or by incubation at 37° C. for 2 hr. After thisincubation, non-specific binding is blocked by incubating the solidsupport with 3% bovine serum albumin in phosphate-buffered saline (PBS)for at least 1 hr at room temperature. The wells are then washed withPBS.

EGF receptors purified as in Example 2, are incubated with the coatedwells for 2 hr at 37° C. in order to allow the EGF receptor to bind theimmobilized compounds. The wells are then washed with PBS.

Bound EGF receptor is detected by incubating the wells with mouseanti-EGF receptor antibodies (Sigma, St. Louis, Mo. or UBI, Lake Placid,N.Y.). After washing the support, the support is incubated withhorseradish peroxidase conjugated to goat anti-mouse secondary antibody(Amersham, Arlington Heights, Ill.). The support is then washed asabove.

Horseradish peroxidase reporter groups are then quantified by incubatingthe support with substrate, as described in Example 3. Candidatecompounds that generate a signal that is at least two-fold abovebackground are considered to bind to EGF receptor.

Example 5 Inhibition of EGF Receptor Autophosphorylation

This Example illustrates the inhibition of immunoprecipitated EGFreceptor autophosphorylation by carbohydrates of this invention.

EGF receptor was immunoprecipitated from detergent solubilized A431cells with a polyclonal anti-EGF receptor antibody coupled to ProteinA-agarose beads. The beads for immunoprecipitation are prepared byincubation of 2-5 μg of a polyclonal anti-EGF receptor antibody (UBI,Lake Placid, N.Y.) with 50 μl of Protein A-agarose beads (Sigma, St.Louis, Mo.) in a total volume of 300 μl PBS for 1 hr at roomtemperature. The armed beads are then incubated with 500 μg to 1.0 mg ofthe proteins from an A431 cell lysate for 16 hr at 4° C. The beads arethen washed three times with PBS and once with phosphorylation buffer.The entire sample is used for one kinase reaction.

In the first experiment, equal portions of the immunoadsorbed EGFreceptor complexes were incubated without carbohydrate, or with either70 or 130 μg of 3′SL-HSA, in a total volume of 250 μl for 1 hr at roomtemperature. 100 ng of EGF was added to the samples, which were thenincubated for 5 minutes in ice. 1 μCi of radiolabeled [γ³²P]ATP wasadded to each sample and, after 20 minutes of incubation on ice, thetubes were centrifuged for 20 seconds in a microfuge, the supernatantremoved, and 20 μl of 2×electrophoresis buffer was added. Samples wereboiled for 2 minutes and loaded on an 8% polyacrylamide gel. Followingelectrophoresis, the gel was dried and autoradiographed, and theradioactivity was quantified using a phosphoimage analyzer (BioRad,Hercules, Calif.).

The level of autophosphorylation, as measured by the amount of ³²Pincorporated into the immunoprecipitated EGF receptor, is shown in FIG.1. These results indicate that 3′SL conjugated to HSA is an effectiveinhibitor of EGF receptor autophosphorylation.

In a second experiment, a series of carbohydrates according to thisinvention were evaluated for the ability to inhibit EGF receptorautophosphorylation. The assays were performed as described above,except that 5 μg of each carbohydrate was used. A dose titration curve(not shown) indicated that the 70 μg used in the first experiment waswell above maximal inhibition, and that (for 3′SL-HSA) 5 μg was withinthe linear range of the inhibition curve.

The level of autophosphorylation, expressed as percent of unstimulatedresponse (i.e., ³²P incorporation in the absence of EGF), is presentedin FIG. 2, where lane 1 shows the response in the absence of EGF, lane 2shows the response in the presence of EGF (without carbohydrate), andlanes 3-6 show the response in the presence of EGF and one of thefollowing: LNnT-HSA, 3′SL-HSA, 6′SL-HSA and LSTa-HSA, respectively. Eachof these conjugates was effective in inhibiting EGF receptorautophosphorylation. Lane 7 also shows the response in the presence of3′SL-HSA, which demonstrates the reproducibility of this assay.

In a third experiment, 3′SL coupled to a dendrimer as described inExample 1 was evaluated for inhibition of EGF receptorautophosphorylation. The assay was performed as described above, using 1or 10 μg of either 3′SL-BSA or 3′SL-dendrimer. The results, presented inFIG. 3, indicate that both 3′SL-BSA and 3′SL-dendrimer inhibitautophosphorylation.

Example 6 Inhibition of EGF Receptor Kinase Activity

This example illustrates the inhibition of EGF receptor kinase activityin cell membranes.

Cell membrane preparations containing EGF receptor were prepared asdescribed in Example 2. Phosphorylation of membrane proteins by membranekinases was carried out as described by Pike et al., Proc. Natl. Acad.Sci USA 79:1443 (1982), using 40 μg of A431 cell membrane preparations.Assays were carried out in a volume of 40 μl containing 20 mM HEPES, pH7.4, 100 mM NaCl, 2 mM MgCl₂, and 100 μM sodium orthovanadate. TritonX-100 solubilized membrane (5 μg) was preincubated with or withoutcarbohydrate (either 3′SL-BSA or 3′SL-dendrimer) for 30 minutes, andthen with 50 ng EGF for 5 minutes prior to initiation of the reaction.The kinase reaction was started by the addition of 10 μM [γ³²P] ATP(1,000-30,000 Ci/mmol) at 4° C., and was allowed to proceed for 5minutes to allow for autophosphorylation. 2 mM RR-src peptide was thenadded. The reaction was allowed to proceed for an additional 10 minutesat 4° C., and was stopped by the addition of 50 μl ice cold 10%trichloroacetic acid. Following centrifugation, the supernatantcontaining the phosphorylated RR-src peptide was transferred ontophosphocellulose paper (Whatman P81, Whatman, Maidstone, England) andwashed three times in 0.075 M phosphoric acid. The dried papers werethen placed into a liquid scintillation counter.

The results, shown in FIG. 4, are expressed as the mean of threereplicates for each data point. Column 1 represents the incorporation of³²P in the absence of EGF, and column 2 shows the incorporation in thepresence of EGF, without carbohydrate. Columns 3 and 4 show theinhibition of incorporation (in the presence of EGF) by 3′SL-BSA and3′SL-dendrimer, respectively.

These results show that both 3′SL-BSA and 3′SL-dendrimer inhibit EGFstimulated kinase activity.

Example 7 Measurement of EGF Receptor Kinase Activity in Intact Cells

Autophosphorylation of the EGF receptor in intact cells is determinedwith anti-phosphotyrosine antibody as described in Pike et al., Proc.Natl. Acad. Sci. USA 79:1443 (1982). Human epidermoid carcinoma cells(A1S) (cultured as described in Hanai et al., J. Biol. Chem. 263:10915(1988)) are seeded in 25 cm² flasks in appropriate media with or withoutcarbohydrate or carbohydrate analog. The cells are cultured for threedays, and then transferred to serum-free media (2 ml) supplemented with100 ng/flask of EGF for 10 minutes at 37° C. After washing twice withice-cold PBS, the monolayer is solubilized with buffer containing 50 mMHEPES (pH 7.4), 150 mM NaCl, 100 mM NaF, 1 mM MgCl₂, 1.5 mM EGTA, 200 μMsodium orthovanadate, 1% Triton X-100, 10% glycerol, 1 mM PMSF, 10 μg/mlaprotinin, 5 mg/ml sodium deoxycholate, and 1 mg/ml sodiumdodecylsulfate. After determination of the protein concentration of thelysates, 50 μg of soluble protein is resolved by SDS-PAGE andtransferred electrophoretically onto nitrocellulose paper (BioRad,Hercules, Calif.). Phosphotyrosine containing proteins are identifiedwith a mouse monoclonal anti-phosphotyrosine antibody PY-20 (ICN, CostaMesa, Calif.) and detected by sequential blotting with biotinylated goatanti-mouse IgG, streptavidin alkaline phosphatase conjugate and BCIP/NBT(ICN).

Example 8 Incorporation of 3′ Sialyllactose Analog into a Liposome

A liposome incorporating a carbohydrate is prepared by mixingcarbohydrate (for example, 3′sialyllactose analog), dipalmitoylphosphatidyl choline, and cholesterol (1:5:3 by weight) inmethanol:chloroform (1:1) and drying the mixture. Phosphate bufferedsaline is added to the mixture and it is sonicated for 30 minutes toproduce liposomes with a range of sizes. Liposomes with a narrow sizedistribution are isolated by filtration through a polycarbonate filterwith an 80 to 100 nm pore size, yielding liposomes with similardiameters.

To incorporate a toxic compound into the liposome, an agent such asdaunomycin is included into the mixture as follows:

A liposome is prepared by mixing carbohydrate, dipalmitoylphosphatidylcholine, cholesterol, and an antioxidant such as Vitamin E(7.5:38:23:1 by weight) in methanol:chloroform (1:1) and drying themixture. 150 mM ammonium sulfate, pH 5.5, is added to the mixture and itis sonicated for 30 minutes to produce liposomes with a range of sizes.Liposomes with a narrow size distribution are isolated by passing thismixture through a polycarbonate filter with 80 to 100 nm pore sizetwenty to thirty times, to yield liposomes with a similar diameter. Theliposomes are then dialyzed with 5% glucose, pH 7, to set up a pHgradient. The daunomycin is incorporated into the liposomes by heatingthe liposomes at 60° C. for one hr with 3.8 M daunomycin in 5% glucose,pH 7. The preparation is cooled to room temperature and unincorporateddaunomycin is removed by ion exchange.

Example 9 Assay for Binding of EGF Receptor to Immobilized 3′SL-HSA

3′SL-HSA is incubated in Falcon probind™ microtiter plate (Plate 1) atserial dilutions in Tris-CA⁺⁺ buffer (0.05M Tris HCl, 0.15M NaCl, 2 mMCaCl₂, pH 7.4). Plate 1 is covered and incubated overnight at 4° C.After incubation, 100 μl/well of 2% BSA in Tris-Ca⁺⁺ buffer is added andincubated at room temperature for 2 hours. This is a minimum time andcan be extended if necessary. During incubation, EGF receptor (Sigmacat#E-1886) is titrated (2×serial dilution) in 1% BSA in Tris-Ca⁺⁺buffer using U-shaped low bind microtiter plates (Plate 2). Seriallydilute up to row 9. Rows 10, 11, and 12 should be just buffer. Finalvolume should be 120 μl/well and the first well should contain 10 μg/mlof EGF receptor. Plate 1 is washed four times with Tris-Ca⁺⁺ buffer inthe automatic plate washer. 100 μl/well is transferred from Plate 2 toPlate 1 starting from lowest concentration of EGF receptor using an8-channel pipettor from right to left. Discard Plate 2. Plate 1 isincubated while rocking at room temperature for 2 hours; and washed fourtimes with Tris-Ca⁺⁺ buffer using automatic plate washer.

100 μl/well of 1/1,000 dilution of anti-EGF receptor antibody (Sigmacat#E-2760) in Tris-Ca⁺⁺ buffer, 1% BSA is added. Incubate while rockingat room temperature for 1 hour. Wash four times with Tris-Ca⁺⁺ bufferusing automatic plate washer. 100 μl/well of peroxidase-labeled goatanti-mouse IgG (KPL labs, cat#074-1806) at 1 μg/ml in Tris-Ca⁺⁺ buffer,1% BSA is added. Incubate while rocking at room temperature for 1 hour.Wash four times with Tris-Ca⁺⁺ buffer using automatic plate washer.

100 μl/well of Substrate (mix TMB reagent and H₂O₂ at 1:1 ratio) isadded with an 8-channel pipettor from right to left. Incubate at roomtemperature for 3 minutes. The reaction is stopped by adding 100 μl/wellof 1M H₃PO₄ using the 8-channel pipettor from right to left. Readabsorbance of light at 450 nm. The results are shown in FIG. 5.

Example 10 Binding of EGF Receptor External Domain to ImmobilizedNeoglycoconjugates

Neoglycoconjugates (LSTb/BSA or 3′SL/dendrimer) were spotted at theamounts indicated on a PVDF membrane (Millipore). The non-specificbinding sites were then blocked with 3% BSA in phosphate-buffered saline(PBS) for 30 min. at room temperature. The PVDF membrane was thenincubated for two hours with A431 cell (human epidermoid carcinoma)culture supernatant enriched in EGF receptor external domain. Afterwashing with PBS, the membrane was then incubated with anti-EGF receptormonoclonal antibody (UBI), directed against the external domain, at a1/1000 dilution for 1 hour at room temperature. The immunocomplex wasthen detected using a secondary antibody linked to horseradishperoxidase and an ECL kit (Amersham). The results are shown in FIG. 6.

The external domain of the EGF receptor was isolated from the cellculture supernatants of A431 cells. This soluble form of the receptorcontaining the EGF binding domain arises from alternate splicing of thefull length message. The material used in the above assay was preparedby ultra filtration of A431 cell culture supernatant to concentrate EGFbinding proteins. This was followed by immunoadsorbtion with anti-EGFreceptor antibodies directed against the cytoplasmic domain of thereceptor in order to remove any full length receptor in culturesupernatant. The presence of the external domain was confirmed bywestern blot.

Example 1 Effect of 3′SL Conjugates on the Proliferation of HumanEpidermoid Carcinoma Cells (A1S)

The effects of 3′SL/BSA and 3′SL/dendrimer were tested for their abilityto inhibit tumor cell growth in culture. For these experiments, 1×10⁴A1S cells were seeded in triplicate onto 24-well plates (Falcon) inDMEM-F12 containing 10% supplemented calf serum. After cell attachmentto the plastic substratum (4-5 hours), the medium was removed andreplaced with DMEM-F12 containing 1% FBS. The 3′SL conjugates were addedat the amount indicated in FIG. 7 per ml when the medium was replaced.The cells were then allowed to grow in culture for 48 hours. The cellswere stimulated with 100 ng/ml EGF for 6 hours prior to addition of[³H]TdR for 18 hours. The data presented in FIG. 7 are reported as theaverage DPM incorporated per triplicate samples. The data presented inthe figure show that the 3′SL conjugates are able to inhibit the EGFdependent cell growth of A1S tumor cells.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually incorporated by reference.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

What is claimed is:
 1. A pharmaceutical composition, comprising: (a) a compound having the formula: X-Y-Z-R wherein X is a sialic acid or a non-saccharide group that mimics the structure of a sialic acid group Y and Z are independently a monosaccharide residue or a non-saccharide group that mimics the structure of a monosaccharide residue, and R is a carrier group, or a carbohydrate compound having a type 2 saccharide chain without a sialic acid residue or an analog thereof, and with a carrier group, wherein the carrier group is a cytotoxic agent, toxin or radionuclide that inhibits tumor cell growth or kills tumor cells; and (b) a pharmaceutically acceptable carrier or diluent.
 2. The composition of claim 1 wherein said compound is incorporated into a liposome.
 3. The composition of claim 1 wherein the compound is 3′-sialyllactose or an analog thereof.
 4. The composition of claim 3 wherein the compound is 3′-sialyllactose.
 5. The composition of claim 3 or 4 wherein said compound is incorporated into a liposome. 